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Nephrocalcinosis is a risk factor for kidney failure in primary hyperoxaluria

2014; Elsevier BV; Volume: 87; Issue: 3 Linguagem: Inglês

10.1038/ki.2014.298

1523-1755

Xiaojing Tang, Eric J. Bergstralh, Ramila A. Mehta, Terri J. Vrtiska, Dawn S. Milliner, John C. Lieske,

Parathyroid Disorders and Treatments

Stone formation and nephrocalcinosis are both very common features of primary hyperoxaluria, yet the extent of each disease varies markedly between patients. Here we studied whether kidney damage from nephrocalcinosis and/or stone related events contributed to end-stage kidney disease (ESKD). Clinical information was analyzed from 348 patients enrolled in the Rare Kidney Stone Consortium Primary Hyperoxaluria registry and included demographic, laboratory and imaging features. Among all patients there were 277 with type 1, 37 with type 2, and 34 with type 3 primary hyperoxaluria. Overall, 58% passed a stone (mean 0.3/year) and one or more urologic procedures were required by 70% of patients (mean 0.15/year). Nephrocalcinosis was found in 34% of patients, including 41% with type 1 primary hyperoxaluria. High urine oxalate was associated with increased risk for both nephrocalcinosis and stone number, while low urine citrate was a risk factor for stone events and stone number. After adjustment for the type of primary hyperoxaluria, diagnosis by family screening and age at first image, the overall adjusted hazard ratio for ESKD among those with a history of nephrocalcinosis was 1.7 [95% CI 1.0–3.0], while the risk was 4.0 [1.9–8.5] for new onset nephrocalcinosis during follow-up. In contrast, the number of stones and stone events were not significantly associated with ESKD risk. Thus, nephrolithiasis and nephrocalcinosis appear to be pathophysiologically distinct entities. The presence of nephrocalcinosis implies increased risk for ESKD. Stone formation and nephrocalcinosis are both very common features of primary hyperoxaluria, yet the extent of each disease varies markedly between patients. Here we studied whether kidney damage from nephrocalcinosis and/or stone related events contributed to end-stage kidney disease (ESKD). Clinical information was analyzed from 348 patients enrolled in the Rare Kidney Stone Consortium Primary Hyperoxaluria registry and included demographic, laboratory and imaging features. Among all patients there were 277 with type 1, 37 with type 2, and 34 with type 3 primary hyperoxaluria. Overall, 58% passed a stone (mean 0.3/year) and one or more urologic procedures were required by 70% of patients (mean 0.15/year). Nephrocalcinosis was found in 34% of patients, including 41% with type 1 primary hyperoxaluria. High urine oxalate was associated with increased risk for both nephrocalcinosis and stone number, while low urine citrate was a risk factor for stone events and stone number. After adjustment for the type of primary hyperoxaluria, diagnosis by family screening and age at first image, the overall adjusted hazard ratio for ESKD among those with a history of nephrocalcinosis was 1.7 [95% CI 1.0–3.0], while the risk was 4.0 [1.9–8.5] for new onset nephrocalcinosis during follow-up. In contrast, the number of stones and stone events were not significantly associated with ESKD risk. Thus, nephrolithiasis and nephrocalcinosis appear to be pathophysiologically distinct entities. The presence of nephrocalcinosis implies increased risk for ESKD. The primary hyperoxalurias (PHs) are autosomal recessive inherited diseases caused by defects in glyoxylate metabolism, resulting in overproduction of oxalate.1.Hoppe B. Beck B.B. Milliner D.S. The primary hyperoxalurias.Kidney Int. 2009; 75: 1264-1271Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar,2.Monico C.G. Rossetti S. Belostotsky R. et al.Primary hyperoxaluria type III gene HOGA1 (formerly DHDPSL) as a possible risk factor for idiopathic calcium oxalate urolithiasis.Clin J Am Soc Nephrol. 2011; 6: 2289-2295Crossref PubMed Scopus (93) Google Scholar Three types of PH have been described to date, each involving a different enzyme within the oxalate metabolic pathways. PH type 1 (PH1) is caused by low or absent activity of liver-specific peroxisomal alanine: glyoxylate aminotransferase (AGT), which results in increased urinary excretion of both oxalate and glycoalate.3.Danpure C.J. Primary hyperoxaluria: from gene defects to designer drugs?.Nephrol Dial Transplant. 2005; 20: 1525-1529Crossref PubMed Scopus (33) Google Scholar In PH type 2 (PH2), deficiency or absence of glyoxylate reductase/hydroxy-pyruvate reductase (GRHPR) leads to elevated urinary excretion of both oxalate and L-glyceric acid.4.Cregeen D.P. Williams E.L. Hulton S. et al.Molecular analysis of the glyoxylate reductase (GRHPR) gene and description of mutations underlying primary hyperoxaluria type 2.Hum Mutat. 2003; 22: 497Crossref PubMed Google Scholar A third type of PH (PH3) was recently found to be due to abnormality of the enzyme 4-hydroxy-2-oxaloglutarate aldolase (HOGA1).2.Monico C.G. Rossetti S. Belostotsky R. et al.Primary hyperoxaluria type III gene HOGA1 (formerly DHDPSL) as a possible risk factor for idiopathic calcium oxalate urolithiasis.Clin J Am Soc Nephrol. 2011; 6: 2289-2295Crossref PubMed Scopus (93) Google Scholar The exact mechanism of oxalate overproduction in PH3 remains to be determined. Humans cannot degrade oxalate which is primarily eliminated through the kidneys as an end product of metabolism. Thus PH is characterized by very high urinary oxalate excretion, typically >1mmol/1.73m2/day. Consequently, tubular fluid becomes supersaturated for calcium oxalate, which can result in the formation of crystals within the lumen. Once formed, crystals can attach to the surface of renal tubular cells. In the medullary collecting duct this can result in tubular plugging and a nidus for stone formation, while in more proximal nephron segments adherent crystals can be endocytosed and/or transcytosed into the renal interstitium leading to nephrocalcinosis (NC). If the glomerular filtration rate (GFR) decreases due to progressive renal damage, oxalate accumulates in the bloodstream and crystals can deposit in the parenchyma of most solid organs, bones, joints and retina once supersaturation is exceeded (∼30μM/l).5.Hoppe B. Kemper M.J. Bokenkamp A. et al.Plasma calcium oxalate supersaturation in children with primary hyperoxaluria and end-stage renal failure.Kidney Int. 1999; 56: 268-274Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar PH often leads to end-stage kidney disease (ESKD) and the death of many patients if untreated. Stone formation and NC are both very common features of PH, yet the numbers and extent vary markedly between individuals. Thus, we investigated whether NC and/or stone related events contributed to ESKD. A total of 379 PH patients were enrolled in the Rare Kidney Stone Consortium (RKSC) registry through April 2013. After excluding 31 unclassified PH patients (25 in whom PH1, PH2, or PH3 were all negative by genetic analysis and 6 historical patients with PH of unknown type), 348 patients were left for further analysis, with 151 (43%) patients known to have ESKD at the time of this analysis. There were 277 (79.6%) PH1, 37 (10.6%) PH2, and 34 (9.8%) PH3 in the registry. Among them 322 (256 PH1, 32 PH2 and 34 PH3) were diagnosed or confirmed by genetic testing or liver biopsy. In the remainder, marked hyperoxaluria in the absence of identifiable secondary cause and in combination with hyperglycolic aciduria provided PH1 diagnosis (n=21) and hyperoxaluria in combination with hyperglyceric aciduria provided PH2 diagnosis (n=5). Family screening identified 29 patients, including 21 with PH1, 7 with PH2 and 1 with PH3. As expected, those identified by family screening were diagnosed at a younger age (4.0 vs. 7.2 yrs, P=0.01), but had a similar risk of NC before ESKD (31.0 vs. 34.0%, P=0.75). Median age at first symptoms and at diagnosis of all patients was 4.9 (1.6–13.3) and 10.4 (4.0–27.9) years old, respectively. Median age at last follow-up was 22.5 (10.0–40.7) years old. Stone burden and imaging results are shown in Table 1. Altogether, 58.4% of patients passed a stone, with a mean of 0.3 passed/yr, translating to about 1stone every 3.3 years. One or more urologic procedures were required by 70.2% of patients ever (mean 0.15 procedures/yr). Clinical features by PH type are in Supplementary Table S1.Table 1Clinical stone events and imaging findings for all PH patientsMean/Median (25th, 75th)No. of patients (%)Clinical stone events (n=348) Stones passed/yr0.30/0.04 (0.00–0.26)121/291 (58.4%) Stone procedures/yr0.15/0.06 (0.00–0.17)172/245 (70.2%) Stone eventsaStone event is defined as any clinical event associated with kidney stones, including stone passage or stone procedure./yr0.42/0.13 (0.00–0.43)212/300 (70.7%)Image findings (n=303) Stones per image before ESKD3.5/2.0 (1.0–4.3)185/303 (61.1%bPercent of imaged subjects (n=303) who ever had a stone on imaging.) NC everNA112/303 (37.0%) NC before ESKDNA79/235 (33.6%)Abbreviations: ESKD, end-stage kidney disease; NA, not applicable; NC, nephrocalcinosis; PH, primary hyperoxaluria.a Stone event is defined as any clinical event associated with kidney stones, including stone passage or stone procedure.b Percent of imaged subjects (n=303) who ever had a stone on imaging. Open table in a new tab Download .doc (.09 MB) Help with doc files Supplementary Tables Abbreviations: ESKD, end-stage kidney disease; NA, not applicable; NC, nephrocalcinosis; PH, primary hyperoxaluria. Kidney imaging was documented in 303 (87%) patients, with a median of 2 tests per patient (1,324 images total); 25.2% were computed tomography (CT) scans, 52.6% ultrasounds (US) and 22.2% kidney/ureter/bladder (KUB) X-rays. NC was defined as a pattern of calcification occurring in the renal parenchyma, most prominent at the corticomedullary juncture, while stones were discrete structures≥1mm in size in the renal papillae or collecting system.6.Habbig S. Beck B.B. Hoppe B. Nephrocalcinosis and urolithiasis in children.Kidney Int. 2011; 80: 1278-1291Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Figure 1a depicts a patient with diffuse NC at the corticomedullary juncture and medulla as well as a few discrete papillary tip stones, while Figure 1b and c show the typical appearance of NC by ultrasound, with increased pyramidal and cortical density. Kidney stone and NC were documented in 61% (185/303) and 37% (112/303) of those with imaging. NC was noted more commonly on US (20%) as opposed to KUB (12%) or CT (11%) (P<0.001). The distribution of imaging method (US/CT/KUB) varied slightly by PH type being 55/26/20% for PH1, 44/18/38% for PH2, and 48/28/24% for PH3 (P<0.001). Among the 303 patients with imaging, 68 had ESKD before first image, leaving 235 with a first image prior to ESKD. In these subjects, 24% (56/235) of patients had both NC and kidney stone, 10% (23/235) had NC only and 47% (111/235) had kidney stone only. NC (prior to ESKD) was found in 69 (40.6%) PH1, 8 (25%) PH2 and 2 (6.1%) PH3 patients, respectively. Table 2 lists a comparison of clinical and laboratory features of patients with and without NC before ESKD for all PH patients and also the PH1 subgroup. Patients with NC did not differ significantly by gender but were diagnosed at a younger age (median 4.4) compared to those without NC (median 9.3). Urinary oxalate was significantly higher in patients with NC, while the 24h urine calcium was significantly lower. NC and kidney stones were not significantly associated. Kidney stones were found in 70.9% of those with NC compared to 71.2% of those without NC (P=0.93). However, those patients with NC had more stones per image than those without (3.0 versus 2.0, P=0.02). NC was more common both at presentation (prevalent) and during follow-up (incident) in PH1 compared to PH2 and PH3 (P<0.001). Trends for NC and stones were similar for PH 2 (Supplementary Table S2). The number of PH3 NC cases was too small to allow for meaningful subanalysis. As shown in Figure 2, NC was prevalent in 30% of PH1 patients on first image, while the cumulative proportion with NC increased to 48% at 10 years and 57% at 20 years of follow-up. In contrast, 32% of PH2 and 7.1% of PH3 patients had NC at 5 years follow-up, and the percentages did not increase further.Table 2Characteristics of all PH patients and the PH1 subgroup with or without nephrocalcinosisPHPH1Without NC (N=156)With NC (N=79)Without NC (N=101)With NC (N=69)Gender Male/Female86/7045/3454/4739/30Age, yrs At diagnosis9.3 (4.5, 24.7)4.4 (1.8, 10.0)*11.5 (5.6, 30.7)4.2 (1.9, 9.4)* At last follow-up22.7 (10.1, 44.7)15.4 (5.8, 23.9)*25.7 (13.2, 44.8)15.1 (5.8, 22.6)*Stone events, /yr0.18 (0.06, 0.47)0.27 (0.00, 0.52)0.16 (0.05, 0.45)0.28 (0.00, 0.59)Stones per image2.0 (0.8, 4.0)3.0 (1.5, 6.1)#2.6 (1.0, 4.0)3.8 (1.7, 6.8)#P[ox], μmol/L4.6 (2.7, 12.0)8.7 (3.7, 17.5)#6.6 (3.0, 15.6)8.8 (4.1, 17.6)U[ox], mmol/24haAdjusted by body surface area; *P<0.01; #P<0.05.1.3 (0.9, 1.9)2.0 (1.3, 2.6)*1.3 (0.9, 2.1)2.0 (1.3, 2.6)*U[Ca], mg/24haAdjusted by body surface area; *P<0.01; #P<0.05.90.2 (59.2, 149.2)63.0 (43.4, 90.9)*79.2 (51.5, 112.7)55.6 (39.9, 85.7)*U[citrate], mg/24haAdjusted by body surface area; *P<0.01; #P<0.05.445 (264, 750)346 (190, 608)336 (210, 643)294 (183, 490)U[volume], L/24haAdjusted by body surface area; *P<0.01; #P<0.05.2.4 (1.9, 3.1)2.8 (2.2, 3.6)2.4 (1.8, 3.2)2.8 (2.3, 3.6)Patients who had ESKD before first image were excluded in the analysis. All plasma and urinary results were the average of all values prior to ESKD. Gender is expressed as number and statistical analysis was made by chi-square. The rest are expressed as median (interquartile range) and statistical analysis was made by rank sum test.a Adjusted by body surface area; *P<0.01; #P<0.05. Open table in a new tab Patients who had ESKD before first image were excluded in the analysis. All plasma and urinary results were the average of all values prior to ESKD. Gender is expressed as number and statistical analysis was made by chi-square. The rest are expressed as median (interquartile range) and statistical analysis was made by rank sum test. There was less evidence that stone-related events associated with laboratory characteristics (Table 3). Urine citrate was inversely correlated with stone passage (Spearman correlation coefficient (r)=-0.22, P=0.004), while the average stone number per image positively associated with plasma and urinary oxalate, and negatively with urine citrate and urine calcium at last follow-up. No plasma or urine measurements correlated with the number of surgical interventions. In the PH1 subgroup, correlations between laboratory characteristics and kidney stone events were similar, although the correlation coefficients of urinary citrate with stone number (r=-0.19, P=0.07) and stone passage (r=-0.18, P=0.06) were of borderline significance in this smaller subset of the total group. Trends for correlation between urine chemistries and stone events were similar when analyzed by PH subtype, although most findings no longer achieved statistical significance in these smaller sample sizes (Supplementary Table S3).Table 3Correlation of stone burden with lab results in all PH patientsStones passed/yrStone procedures/yrStone events/yrMean stone number/imagePlasma oxalate (μmol/l) r-0.03-0.02-0.060.31 P0.690.840.520.001U[ox]mmol/1.73m2 /24h r-0.090.03-0.050.16 P0.190.650.460.05U[Ca] mg/1.73m2/24h r0.050.100.08-0.26 P0.540.220.290.002U[citrate] mg/1.73m2/24h r-0.22-0.02-0.15-0.35 P0.0040.820.05<0.001U[volume]ml/1.73m2/24h r-0.020.02-0.040.06 P0.730.840.550.46Current eGFR ml/min/1.73m2 r-0.050.100.03-0.35 P0.430.180.70<0.001eGFR decline rate ml/min/1.73m2/yr r-0.040.04-0.01-0.13 P0.620.610.920.13P: P value; r: correlation coefficients of Spearman rank correlation. Open table in a new tab P: P value; r: correlation coefficients of Spearman rank correlation. The impact of NC and other factors on renal outcome was studied in the 235 subjects without ESKD prior to their first image. Altogether, 57 (56 PH1, 1 PH2, 0 PH3) of 235 patients developed ESKD after the first image. Table 4 summarizes the effect of NC on ESKD for all PH patients and the PH1 subgroup separately. The ESKD hazard ratio (HR) among all PH patients with NC-ever was 2.1 [95% confidence interval (CI) 1.2–3.6]. After adjustment for PH type, PH diagnosis by family screening, and age at first image, the overall adjusted HR of ESKD among those with NC ever was 1.7 [1.0–3.0]. The effect on ESKD risk was larger for new onset NC during follow-up (HR 4.0 [1.9–8.5]) as opposed to NC at first image (HR 1.2 [0.6–2.3]). The effect of NC on ESKD risk in the PH1 subgroup was similar with a HR of 1.5 [0.9–2.5] and 3.6 [1.7–7.7] for NC-ever and new onset NC, respectively. After adjustment for baseline urinary oxalate, new onset NC remained a significant risk factor for ESKD in all PH patients (HR 4.3 [1.6–12]) and in PH1 (HR 3.4 [1.2–10]).Table 4Risk of ESKD among PH patients with and without NCGroup of PH patientsHazard ratio (95% CI)Total NCPrevalent NCIncident NCbPatients with prevalent NC are those who found NC on first image. Patients with incident NC are those who had new NC during follow-up.All PH types (N=235, 57 ESKD) No adjustment2.1 (1.2–3.6)1.5 (0.8–2.8)5.1 (2.4–11.0) Adjusting PH1, patients by family screening and age on 1st image1.7 (1.0–3.0)1.2 (0.6–2.3)4.0 (1.9–8.5) Adjusting PH1 and stone numbers on 1st imageaStone number is available for 140 PH patients with 25 ESKD events overall, and for 94 PH1 patients with 24 ESKD events.3.9 (1.7–8.9)1.6 (0.4–5.7)8.2 (3.2–21.3)PH1 subgroup (N=170, 56 ESKD) No adjustment1.5 (0.9–2.5)1.1 (0.6–2.0)3.6 (1.7–7.7) Adjusting patients by family screening and age on 1st image1.6 (0.9–2.9)1.2 (0.6–2.3)3.6 (1.6–7.8) Adjusting stone numbers on 1st imageaStone number is available for 140 PH patients with 25 ESKD events overall, and for 94 PH1 patients with 24 ESKD events.3.5 (1.5–8.3)1.6 (0.4–5.7)6.9 (2.6–19.0)Patients who had ESKD before first image were excluded in the analysis.a Stone number is available for 140 PH patients with 25 ESKD events overall, and for 94 PH1 patients with 24 ESKD events.b Patients with prevalent NC are those who found NC on first image. Patients with incident NC are those who had new NC during follow-up. Open table in a new tab Patients who had ESKD before first image were excluded in the analysis. When PH patients were subdivided into three groups according to the presence (prevalent) or absence of NC on the baseline and follow-up images, the decline in eGFR was more significant in those with incident NC on the follow-up images (Table 5). The trend and magnitude of kidney function loss in relation to the presence of NC were similar in the PH1 subgroup; median eGFR decline rate among those without NC, with prevalent NC, and with incident NC were -0.25, -0.15 and -1.46ml/min/1.73m2/yr, respectively (P=0.16). Figure 3 graphically illustrates the cumulative ESKD incidence for all PH patients and the PH1 subgroup divided into the three groups (without NC, prevalent NC, incident NC).Table 5Kidney function among PH patients without NC, with NC on first image and with NC during follow-upWithout NCPrevalent NCIncident NCP valueAll PH type1565722 eGFR at diagnosis74.0 (57.6, 97.5)68.2 (50.0, 87.4)69.8 (56.7, 91.7)0.24 eGFR at last follow-upaData were cut at eGFR of 20ml/min/1.73m2.79.8 (55.6, 104.4)69.1 (48.7, 91.9)62.6 (30.4, 89.5)0.03 eGFR decline rate/yr-0.4 (-1.9, 3.0)-0.4 (-2.3, 1.9)-1.2 (-3.8, 0.02)0.08 ESKD21.2%24.6%45.5%0.04PH1 subgroup1015118 eGFR at diagnosis68.9 (51.8, 89.0)64.2 (46.8, 85.3)69.8 (56.7, 90.2)0.56 eGFR at last follow-upaData were cut at eGFR of 20ml/min/1.73m2.67.3 (44.2, 86.9)64.2 (34.5, 91.7)54.9 (28.5, 85.3)0.33 eGFR decline rate/yr-0.3 (-2.5, 1.8)-0.2 (-2.1, 2.8)-1.5 (-3.8, -0.1)0.16 ESKD32.7%27.5%50.0%0.21Patients who had ESKD before first image were excluded in the analysis. All eGFR values were expressed as median (interquartile range).a Data were cut at eGFR of 20ml/min/1.73m2. Open table in a new tab Patients who had ESKD before first image were excluded in the analysis. All eGFR values were expressed as median (interquartile range). Among 170 PH1 subjects with imaging prior to ESKD, 133 had genetic testing for G170R, which appears to confer complete or partial response to pyridoxine responsiveness.7.Monico C.G. Rossetti S. Olson J.B. et al.Pyridoxine effect in type I primary hyperoxaluria is associated with the most common mutant allele.Kidney Int. 2005; 67: 1704-1709Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar Of these, 69 had at least one copy of the G170R AGXT genetic change and 64 did not. The rate of NC was not significantly (P=0.29) different between patients with (32% NC) or without (41% NC) a G170R change. Among the 133 PH1 subjects with imaging prior to ESKD, the effect of a G170R change on ESKD was somewhat protective (HR 0.6[0.3–1.0]) when adjusted for NC. Furthermore, incident NC remained significant in this model (HR 3.7 [1.6–8.9]) when adjusted for G170R status. Conversely, the number of stones per image was not significantly associated with ESKD risk (HR 1.07 [0.98–1.17]). Neither the number of stone events nor stones on imaging was associated with the eGFR decline rate (Table 3). Furthermore, the risk of ESKD among PH patients with NC remained increased (HR 3.9 [1.7–8.9]) after adjustment for stone numbers. NC refers to the deposition of radiologically dense calcium salts in the kidney parenchyma (Figure 1a), and is different from crystal deposition in the collecting ducts or collecting system, which typically leads to kidney stones (nephrolithiasis). It is generally accepted that kidney stones and NC are early and common clinical manifestations of PH,8.Hoppe B. Latta K. von Schnakenburg C. et al.Primary hyperoxaluria—the German experience.Am J Nephrol. 2005; 25: 276-281Crossref PubMed Scopus (57) Google Scholar and our study confirmed that impression in a large cohort with good longitudinal follow-up. Results demonstrate that 61% of patients had kidney stones by imaging and 58% passed a stone during follow-up, averaging about 1 stone every 3.3 years. NC was slightly less common, being present in 37% of patients ever. Although stones were more common than NC, the latter portended an increased risk of ESKD, while the number of stones did not correlate with kidney function over time. Notably, the percentage of PH patients affected by NC and/or stones could have been underestimated, since we assumed that they were not present if not specifically included in the radiologic reports entered into the registry. It is also possible that the incidence of NC could have been underestimated because of a low sensitivity of radiological imaging techniques.9.Cheidde L. Ajzen S.A. Tamer Langen C.H. et al.A critical appraisal of the radiological evaluation of nephrocalcinosis.Nephron Clin Pract. 2007; 106: c119-c124Crossref PubMed Scopus (19) Google Scholar Recent research has demonstrated that ultrasound is a more accurate technique for evaluation of mild to moderate NC in patients with hypoparathyroidism.10.Boyce A.M. Shawker T.H. Hill S.C. et al.Ultrasound is superior to computed tomography for assessment of medullary nephrocalcinosis in hypoparathyroidism.J Clin Endocrinol Metab. 2013; 98: 989-994Crossref PubMed Scopus (33) Google Scholar However, CT remains a superior technique for evaluation of renal stone disease.11.Viprakasit D.P. Sawyer M.D. Herrell S.D. et al.Limitations of ultrasonography in the evaluation of urolithiasis: a correlation with computed tomography.J Endourol. 2012; 26: 209-213Crossref PubMed Scopus (37) Google Scholar Therefore, while the most accurate imaging to evaluate PH patients remains unknown, the lower cost and lack of radiation suggests ultrasound can be useful. PH patients often present with kidney stones, hence the diagnosis can be confused with the far more common idiopathic calcium oxalate stones. Previous studies report the diagnosis of PH was made 1–4 years after the first manifestation, and a high percentage of patients were only diagnosed in ESKD.8.Hoppe B. Latta K. von Schnakenburg C. et al.Primary hyperoxaluria—the German experience.Am J Nephrol. 2005; 25: 276-281Crossref PubMed Scopus (57) Google Scholar,12.Hoppe B. Langman C.B. A United States survey on diagnosis, treatment, and outcome of primary hyperoxaluria.Pediatr Nephrol. 2003; 18: 986-991Crossref PubMed Scopus (157) Google Scholar,13.Takayama T. Nagata M. Ichiyama A. et al.Primary hyperoxaluria type 1 in Japan.Am J Nephrol. 2005; 25: 297-302Crossref PubMed Scopus (23) Google Scholar Our data also revealed a 5 year delay in the diagnosis, while 43% of patients reached ESKD by the last follow-up. Therefore, more effort is needed to shorten the interval between first symptom and diagnosis in order to improve renal survival. Our study suggests that increased urinary oxalate excretion is crucial for the development of both NC and the number of kidney stones on imaging, although no evident interaction was found between urine parameters and clinical stone events (stone passage and stone procedures). This finding was consistent with observations in hyperoxaluric animal models in which NC is associated with increased urinary supersaturation.14.Khan S.R. Nephrocalcinosis in animal models with and without stones.Urol Res. 2010; 38: 429-438Crossref PubMed Scopus (57) Google Scholar Furthermore, in rodent models large crystals can plug terminal collecting ducts and anchor larger stones. It is less clear if collecting duct plugs can anchor stones in humans. Nevertheless, our results stress the importance of an aggressive treatment program to decrease urine calcium oxalate supersaturation and crystallization, with a goal to prevent NC, kidney stones, and possibly ESKD. Citrate supplements are thought to reduce calcium oxalate stone formation in patients with PH.15.Edvardsson V.O. Goldfarb D.S. Lieske J.C. et al.Hereditary causes of kidney stones and chronic kidney disease.Pediatric nephrology. 2013; 28: 1923-1942Crossref PubMed Scopus (155) Google Scholar Several studies also demonstrate that hypocitraturia is a risk factor for NC in preterm neonates, the post transplanted kidney of children, and Dent disease.16.Cebotaru V. Kaul S. Devuyst O. et al.High citrate diet delays progression of renal insufficiency in the ClC-5 knockout mouse model of Dent's disease.Kidney Int. 2005; 68: 642-652Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 17.Stapenhorst L. Sassen R. Beck B. et al.Hypocitraturia as a risk factor for nephrocalcinosis after kidney transplantation.Pediatr Nephrol. 2005; 20: 652-656Crossref PubMed Scopus (48) Google Scholar, 18.Sikora P. Roth B. Kribs A. et al.Hypocitraturia is one of the major risk factors for nephrocalcinosis in very low birth weight (VLBW) infants.Kidney Int. 2003; 63: 2194-2199Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar However, in the current study we did not detect an effect of urinary citrate levels on NC risk among PH patients. Hypocitraturia did associate with an increased number of stones on imaging and clinical stone events. One potential explanation could lie in the presumed site of action of citrate. The formation of calcium-citrate complexes may be more important to prevent calcium oxalate crystallization and tubular plugging in the distal tubule, a presumed precursor of stone formation. Conversely, nephrocalcinosis may involve other processes within proximal tubular cells or the renal interstitium. Indeed, the fact that clinical features associated with NC and stones appear to differ supports the hypothesis that these are two distinct pathophysiological entities. NC correlated with increased risk of subsequent ESKD, while stone events and stone numbers did not, indicating that the ultimate renal consequences of NC and stones are different in PH patients. The role of NC in the destruction of renal parenchyma is still uncertain. Cell culture and animal models have provided evidence that oxalate and calcium oxalate (CaOx) crystals may be injurious to renal tubular cells by both direct and indirect mechanisms, inducing proliferation and/or cell death.19.Scheid C. Koul H. Hill W.A. et al.Oxalate toxicity in LLC-PK1 cells: role of free radicals.Kidney Int. 1996; 49: 413-419Abstract Full Text PDF PubMed Scopus (207) Google Scholar, 20.Verkoelen C.F. Verhulst A. Proposed mechanisms in renal tubular crystal retention.Kidney Int. 2007; 72: 13-18Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 21.Asselman M. Calcium Oxalate Crystal Adherence to Hyaluronan-, Osteopontin-, and CD44-Expressing Injured/Regenerating Tubular Epithelial Cells in Rat Kidneys.J Am Soc Nephrol. 2003; 14: 3155-3166Crossref PubMed Scopus (168) Google Scholar Cell culture studies suggest that the response of renal tubular cells is nephron segment-specific, since CaOx crystals are relatively non-toxic to cells obtained from segments of the nephron where their presence is common (collecting ducts), whereas they cause proliferation in low amounts and cell death in high amounts when proximal tubular cells are exposed.20.Verkoelen C.F. Verhulst A. Proposed mechanisms in renal tubular crystal retention.Kidney Int. 2007; 72: 13-18Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar In markedly hyperoxaluric animals CaOx crystals deposit mainly inside tubules and lead to a lesion mimicking NC in the proximal tubule, associating with cellular injury, inflammation and regeneration.14.Khan S.R. Nephrocalcinosis in animal models with and without stones.